This invention relates to novel cytotoxic macrolide compounds. More particularly, this invention is directed to derivatives of brefeldin A, pharmaceutical formulations comprising said derivatives, and a method of using certain of those derivatives as brefeldin A prodrugs.
Brefeldin A is a macrolide antibiotic first isolated from the fungus Penicillium decumbers. The bicyclic ring structure was subsequently established by X-ray crystallography. Brefeldin A possesses a number of biological properties of potential therapeutic interest, including antitumor, antiviral, antifungal, nematocidal, and antimitotic effects. Mode of action studies have revealed that brefeldin A inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus, causes reversible disassembly of the Golgi complex, and blocks protein transport beyond the Golgi complex. Recently, it has been shown that brefeldin A induces DNA fragmentation that is associated with apoptosis in cancer cells. This recent discovery has stimulated a great deal of interest in the preclinical development of brefeldin A as an anticancer agent.
Clinical use of brefeldin A is severely limited by certain of its pharmacokinetic properties; negligible bioavailability after oral administration, and rapid clearance from the blood plasma after intravenous administration. Studies in Chinese hamster ovary cells have indicated that brefeldin A is secreted as glutathione and cysteine conjugates. Studies have also revealed that glutathione-S-transferase system may be responsible for the inactivation of brefeldin A in mammalian cells.
Formulation of brefeldin A is also complicated by its low solubility in aqueous solutions. Despite the existence of the lactone system and two hydroxyl groups on its bicyclic ring system, brefeldin A is only marginally soluble in aqueous medium. This severely limits the formulation of brefeldin A in solution for intravenous or intramuscular injection.
In accordance with this invention cytotoxic derivatives of brefeldin A having increased solubility and apparent prodrug activity have been prepared. Thus in accordance with one embodiment of this invention there is provided a compound of formula 
wherein R1 and R1xe2x80x2 are independently hydrogen or carboxy substituted C1-C5, alkanoyl, Y is H or OH, and Z is OH or xe2x80x94S(O)nR wherein n is 0, 1 or 2 and wherein R is C1-C6 alkyl, phenyl, or C1-C6 alkyl or phenyl substituted with one or more groups selected from the group consisting of OH, C1-C4 alkoxy, halo, carboxy, carbo(C1-C4 alkoxy), amino, xe2x80x94SO3H, and mono or di (C1-C4 alkyl)amino, provided that when n is 0, R is not a 2-amino-2-carboxy alkyl group or an acylated derivative thereof, and provided that when Y is OH, Z is OH. The compounds of formula I wherein n is 0, 1, or 2 represent the corresponding sulfides, sulfoxide and sulfones, respectively.
This invention also directed to a method for preparing compounds of formula I by reacting brefeldin A, or a derivative thereof, with a thiol of the formula RSH to produce a compound of formula I wherein n is 0. The corresponding compounds wherein n is 1 or 2 are prepared by oxidizing the sulfide intermediates (n=O). In addition, brefeldin A and its derivatives can be optionally reacted with a C1-C6 cyclic anhydride to form a compounds of formula I wherein at least one of R1 and R1xe2x80x2 is carboxy substituted C1-C5 alkanoyl.
In yet another embodiment of this invention there is provided a method for providing therapeutically effective serum levels of brefeldin A in a patient in need of the therapeutic benefit of brefeldin A by administering an effective amount of a compound of formula 1 wherein n is 0 or 1 in a pharmaceutically acceptable carrier.
The present invention further provides pharmaceutical formulations comprising an effective amount of the brefeldin A derivatives for treating a patient having a tumor or other neoplastic disease. As used herein, an effective amount of the brefeldin A derivative defined as the amount of the compound which, upon administration to a patient, inhibits growth of tumor cells, kills malignant cells, reduces the volume or size of the tumors or eliminates the tumor entirely in the treated patient.
The effective amount to be administered to a patient is typically based on body surface area, patient weight, and patient condition. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, E. J., et al., Cancer Chemother. Rep., 50 (4): 219 (1966). Body surface area may be approximately determined from patient height and weight (see e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, pages 537-538 (1970)). An effective amount of the brefeldin A derivative in the present invention can range from about 5 mg/kg to about 100 mg/kg, more preferably from about 0.25 mg/kg to about 50 mg/kg, and most preferably about 0.1 to about 10 mg/kg.
Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage and the possibility of co-usage with other therapeutic treatments including other anti-tumor agents, and radiation therapy.
The pharmaceutical formulation may be administered via the parenteral route, including subcutaneously, intraperitoneally, intramuscularly and intravenously. Examples of parenteral dosage forms include aqueous solutions of the active agent, in a isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carrier. In one preferred aspect of the present embodiment, the brefeldin A derivative is dissolved in a saline solution containing 5% of dinmethyl sulfoxide and 10% Cremphor EL (Sigma Chemical Company). Additional solubilizing agents such as cyclodextrins, which form specific, more soluble complexes with the present compounds, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the present compounds.
The present compound can also be formulated into dosage forms for other routes of administration utilizing well-known methods. The pharmaceutical compositions can be formulated, for example, in dosage forms for oral administration in a capsule, a gel seal or a tablet. Capsules may comprise any well-known pharmaceutically acceptable material such as gelatin or cellulose derivatives. Tablets may be formulated in accordance with conventional procedure by compressing mixtures of the active brefeldin A derivatives and solid carriers, and lubricants well-known to those familiar with the art. Examples of solid carriers include starch, sugar, bentonite. The compounds of the present invention can also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder and conventional fillers and tableting agents.
The following description is provided to illustrate various embodiments of Applicants"" invention, and are not intended to in any way limit the scope of the invention as set forth in this specification and appended claims.
Brefeldin A is a macrolide antibiotic first isolated from the fungus Penicillium decumbens. The structure 1 was subsequently established by X-ray crystallography. It has been known for a long time that brefeldin A possesses a number of interesting biological properties of potential therapeutic interest, including antitumor, antiviral, antifungal, nematocidal, and antimitotic effects. Studies of the mode of action of brefeldin A have revealed that it inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus, causes reversible disassembly of the Golgi complex, and blocks protein transport beyond the Golgi complex. In addition, the ability of brefeldin A to induce DNA fragmentation associated with apoptosis in cancer cells has stimulated a great deal of recent interest in its preclinical development as an anticancer agent. However, the potential clinical use of brefeldin A is severely limited by its undesirable pharmacokinetic properties, including negligible bioavailability after oral administration and rapid clearance from the blood plasma after intravenous administration. Formulation of brefeldin A is also complicated by its low aqueous solubility. In view of these problems, additional work in the area of brefeldin A congener and prodrug synthesis is indicated.
Brefeldin A prodrugs would ideally be water soluble compounds which would be readily absorbed after oral administration and would be metabolized back to brefeldin A after systemic distribution in the blood plasma. The Michael addition of thiols to the xcex1,xcex2-unsaturated lactone system present in brefeldin A has been investigated. The resulting sulfides might then be metabolized to sulfoxides after absorption, and the sulfoxides could then conceivably undergo syn elimination back to the xcex1,xcex2-unsaturated lactone system present in brefeldin A. In accordance with this invention, a variety of thiols have been reacted with brefeldin A, and the resulting sulfides have ben oxidized to sulfoxides/sulfones. The cytotoxicities of the resulting compound have been investigated in human cancer cell cultures.
Thiol addition products 2-12 were prepared by reacting brefeldin A with the corresponding thiols in ethanol in the presence of xe2x80x9cproton sponge(copyright)xe2x80x9d [1,8-bis(dimethylamino)naphthalene]. The reactions occurred readily and were found to be highly diastereoselective. The R configuration at C-3 in these products was assigned on the basis of the X-ray structure of a crystalline bis(3,5-dinitrobenzoate) derivative of the adduct formed from brefeldin A and 2-mercaptoethanol. The X-ray and NMR data indicate that there is a conformational change in the macrocycle in going from brefeldin A to the adducts. In brefeldin A, C-2 and C-5 are anti, whereas in the products, they are gauche. The specific thiol addition products prepared were chosen to incorporate a variety of polar, acidic, and basic functional groups that would impart additional aqueous solubility. Compounds 11 and 12 were synthesized because studies in Chinese hamster ovary cells have indicated that brefeldin A is secreted as these glutathione and cysteine conjugates. The biological activities of these two compounds is of interest because of the possibility that the glutathione-S-transferase system is responsible for the inactivation of the antibiotic in mammalian cells. Nine of the sulfides were oxidized to the corresponding sulfoxides 13-21.
Certain mono- and diesters of brefeldin A with polar groups in the side chain were synthesized by the reaction of brefeldin A with succinic anhydride and glutaric anhydride. These succinate and glutarate derivatives might also act as brefeldin A prodrugs and be hydrolyzed back to brefeldin A by esterases present in the blood plasma. The monosuccinate 22 of brefeldin A was obtained by the reaction of 1 with 1.5 equivalents of succinic anhydride in pyridine at 60xc2x0 C. However, when glutaric anhydride was employed using this method, there was no product formation. In order to force the reaction, 4-dimethylaminopyridine was added to the reaction mixture. This led to the formation of both the monoester 23 and the diester 25 along with unreacted starting material. The diester 25 could be separated from the mixture 
by column chromatography on silica gel, but the monoester 23 co-eluted with some impurities and brefeldin A. The reaction was repeated again with 3 equivalents of glutaric anhydride and 2 equivalents of DMAP to afford the desired diester 25 as the major product. A single recrystallization from hexanes and ethyl acetate afforded a pure sample of the diglutarate 25. This method was then applied to the synthesis of the disuccinate 24 of brefeldin A. Reaction of 1 with 3 equivalents of succinic anhydride in the presence of 2 equivalents of 4-dimethylaminopyridine in pyridine at 60xc2x0 C. afforded the desired compound 24 in moderate yield after column chromatographic purification on silica gel and recrystallization.
In order to synthesize and isolate the monoglutarate 23 in pure form, the reaction was attempted in pyridine as the solvent. Since addition of 4-dimethylaminopyridine causes the formation of the diester 25 and the reaction does not occur at 60xc2x0 C., the reaction temperature was raised to 110xc2x0 C. for 36 hours and 4-dimethylaminopyridine was omitted. The TLC of the reaction mixture indicated the formation of the monoglutarate 23 as the major product along with some unreacted starting material. A very minor amount of the undesired diester 25 was also present. Column chromatography on silica gel followed by recrystallization from diethyl ether and hexanes afforded the desired compound 23.
An attempt was also made to introduce additional hydroxyl groups into the brefeldin A system in order to increase aqueous solubility and to provide additional structure-activity information. To this end, the reaction of brefeldin A with osmium tetroxide was investigated. Thus, the reaction of (+)-brefeldin A with two equivalents of N-methylmorpholine oxide (NMO) and a catalytic amount of osmium tetraoxide (OsO4) in a mixture of t-BuOH:H2O was attempted at room temperature. After 4 hours stirring at room temperature the formation of a product was observed on TLC. After usual work up and purification, the dihydroxylation product 26 was isolated in 89% yield. 1H NMR analysis of the reaction product 26 showed disappearance of C-2 and C-3 olefinic protons from 1 and presence of C-10 and C-11 olefinic protons, indicating a regioselective dihydroxylation. The stereochemistry of the dihydroxylation product 26 at C-2 and C-3 has not been determined.
Some attention has also been directed toward the determination of the structures of the reaction products obtained from the oxidation of the methyl 
thioglycolate addition product 7 under forcing conditions. The oxidation of 7 with 1.1 equivalents of m-chloroperbenzoic acid in methylene chloride at 0xc2x0 C. for 2 minutes afforded the corresponding sulfoxide 16 in good yield. However, the treatment of sulfide 7 with 2.2 equivalents of m-chloroperbenzoic acid in methylene chloride at room temperature for 4 hours gave the desired sulfone 27 in 46% isolated yield. When the sulfide 7 was treated with 4.4 equivalents of m-chloroperbenzoic acid in methylene chloride for 7 hours, the sulfur was oxidized to the sulfone and the C-10, C-11 double bond was also oxidized, resulting in compound 28. The stereochemistry of the epoxide has not yet been determined.
The new synthesized brefeldin A prodrug candidates and analogs were examined for antiproliferative activity against human cancer cell lines in the National Cancer Institute screen, in which the activity of each compound was evaluated with approximately 55 different cancer cell lines of diverse tumor origins. The GI50 values obtained with selected cell lines, along with the mean graph midpoint (MGM) values, are summarized in Table 1. The MGM is based on a calculation of the average GI50 for all of the cell lines tested (approximately 55) in which GI50 values below and above the test range (10xe2x88x924 to 10xe2x88x928 molar) are taken as the minimum (10xe2x88x928 molar) and maximum (10xe2x88x924 molar) drug concentrations used in the screening test.
It is apparent from the data in Table 1 as well as from the more extensive data in approximately 55 cell lines (data not shown) that neither brefeldin A nor any of the new derivatives prepared in the present study have significant selectivity for any particular subpanel of cancer cell lines. It is also clear that the sulfide derivatives 2-12 (MGM 0.37-42 xcexcM) are in general much less active that brefeldin A (MGM 0.040 xcexcM). The xcex1,xcex2-unsaturated lactone moiety therefore appears to be an important structural determinant for cytotoxicity. This point is also borne out by the cytotoxic activity of 26 (MGM 3.0 xcexcM) relative to 1 (MGM 0.041 xcexcM), and it is also consistent with a recent report documenting the importance of the xcex1,xcex2-unsaturated lactone moiety for induction of apoptotic DNA fragmentation.
The more active of the sulfide derivatives were 12 (MGM 0.37 xcexcM), 4 (MGM 0.68 xcexcM), 11 (MGM 1.8 xcexcM), and 9 (MGM 2.5 xcexcM). It is worth noting that all of these more active sulfides have side chains containing basic amino groups that could possibly catalyze the elimination of the sulfide to regenerate brefeldin A.
Included in this list are the brefeldin A secondary metabolites 11 and 12, assuming the metabolites in fact have the same configuration as the major thiol addition products.
Turning to the sulfoxides 13-21, it is evident that these compounds are the most active set investigated, with MGM values ranging from 0.037 xcexcM for compound 14 to 0.68 xcexcM for compound 16. The cytotoxicity of 14 is essentially equipotant with that of brefeldin A (MGM 0.040 xcexcM). The average MGM value for the set of sulfoxides 13-21 is 0.24 xcexcM, which is much lower than the 14 xcexcM value calculated for the sulfides 2-12. It seems likely that the sulfoxides undergo elimination during cytotoxicity testing and that the observed biological results are at least in part due to the presence of brefeldin A. This would account for the difference in activity seen between the sulfides and sulfoxides.
In order to investigate the proposed sulfoxide elimination reaction to regenerate brefeldin A, compounds 13, 15, and 20 were placed in a D2O/CD3OD buffer, pH 7.4, containing sodium bicarbonate and sodium acetate at room temperature and the 1H NMR spectra were recorded at various intervals. The conversion of each of these sulfoxides to brefeldin A was monitored by observing the disappearance of the C-2 protons in the starting material and the appearance of the C-2 and C-3 alkene protons in brefeldin A. During this process, no other compounds beside these sulfoxides and brefeldin A were detected. The half-life for the conversion of the sulfoxide 13 to brefeldin A was 87.4 hours, while those of 15 and 20 were 4.81 and 1.87 hours, respectively. It is therefore clear that there is likely to be substantial conversion of the sulfoxides to regenerate brefeldin A during the in vitro cell culture cytotoxicity experiments, especially when one considers that the cull cultures are incubated at 37xc2x0 C. and the present kinetics experiments were performed at 23xc2x0 C. It is also evident that the sulfoxide 20 with the fastest rate of elimination contains a basic dimethylanino group which could facilitate the conversion through deprotonation of one of the C-2 protons xcex1 to the carbonyl.
To determine whether or not the conversion of brefeldin to various sulfide derivatives in fact has an appreciable effect on their solubilities, saturated solutions of brefeldin A and several of the derivatives were prepared in distilled water at room temperature and the concentrations were determined gravimetrically after evaporation of all of the water by azeotropic distillation with ethanol. The solubility of brefeldin A determined in this way was 2.8 mg/mL, while those of the sulfides were: 4, 10 mg/mL; 5, 12 mg/mL; 6, 40 mg/mL; and 11, 35 mg/mL. The increased solubilities of these derivatives will facilitate their formulations for biological evaluation.
The succinates and glutarates 22-25 ranged from being moderately cytotoxic to essentially inactive, and there was a difference in activity between the monoacylated and diacylated products. Of these four compounds, the monosuccinate 22 was the most active, displaying an MGM value of 2.5 xcexcM. This was followed by the monoglutarate 23 (MGM 13 xcexcM). The disuccinate 24 and diglutarate 25 were much less active, displaying MGMs of 89 xcexcM and 98 xcexcM, respectively.
The effect of sulfone vs. sulfoxide substitution can be seen by comparing the activities of 27 (MGM 3.5 xcexcM) and 16 (MGM 0.68 xcexcM). In this particular case, the sulfoxide is more cytotoxic. Oxidation of the C-10 to C-11 double bond in 27 to form 28 (MGM 31 xcexcM) resulted in a significant loss of activity. This is consistent with the observation that epoxidation of the C-10 to C-11 double bond of brefeldin A results in a substantial loss of potency for induction of apoptotic DNA fragmentation.
Overall, the increase in biological activity seen with the conversion of the sulfides to the sulfoxides, along with the documented elimination of several of the sulfoxides to regenerate brefeldin A, provides strong support for the general strategy of employing the sulfide products formed from the addition of thiols to brefeldin A as prodrug candidates. Sulfides attached to acidic or basic functional groups appear to be particularly attractive, since these compounds can be converted to salts having increased solubility in an aqueous environment. Several of the more active brefeldin A derivatives are presently being evaluated in vivo in animal models as anticancer agents.